Amplicon Mapping and Expression Profiling Identify the Fas-Associated Death Domain Gene as a New Driver in the 11q13.3 Amplicon in Laryngeal/Pharyngeal Cancer

Abstract

Purpose: Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene. Experimental Design: To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon. Results: Using array-based comparative genomic hybridization analysis, we detected a region of f1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser 194 phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser 194 phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest. Conclusion: Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit fromTaxol-based chemoradiotherapy.

Cortactin expression predicts poor survival in laryngeal carcinoma

“…For the immunostaining of cortactin we used a commercially available antibody (clone 30/cortactin from BD Transduction Laboratories, Franklin Lakes, NJ, USA) that recognizes all cortactin isoforms and can be applied on archival material. IHC for FADD on the larger series of 167 LSCC was reported earlier (Gibcus et al, 2007b). Ki67 expression was used as a marker for cell proliferation.…”

“…consistently overexpressed genes in the 11q13 amplicon (Gibcus et al, 2007b) were not investigated further because no antibodies were available (TAOS1 and FLJ42258) or appeared to be inappropriate for immunohistochemistry on archive material (PPFIA1 and FGF19) (data not shown). Firstly, we determined whether there was a link between amplification and protein expression.…”

“…We concluded that the selection for tumour cells with the 11q13.3 amplicon during tumorigenesis could be based on the increased doses of one or more of these genes. We proposed FADD as a possible candidate for this selection, since FADD showed the best correlation between DNA amplification and increased expression (Gibcus et al, 2007b). Furthermore, FADD protein expression correlated with disease specific mortality (DSM) in a series of late stage laryngeal carcinomas.…”

“…The intermingled lymphocytes served as a negative control. For FADD the intensity of the positively stained tumour cells was scored and divided into two different categories; low expressors (À, þ ) and high expressors ( þ þ , þ þ þ ) as described previously (Gibcus et al, 2007b).Antibodies against PPFIA1 (LIP.1) (Serra-Pages et al, 1995) and FGF19 (mab969; R&D Systems, Minneapolis, USA) appeared to be inappropriate for IHC analysis (data not shown) and antibodies against FLJ42258 and TAOS1 are presently not available. …”

“…It is believed that amplification increases gene dosage and expression of genes within the amplified region (amplicon) (Albertson, 2006). Recently, we reported that the commonly amplified region is located at 11q13.3 and contains at least six genes (cyclin D1, TAOS1, FGF19, FADD, PPFIA1 and cortactin) that are overexpressed when amplified (Gibcus et al, 2007b). We concluded that the selection for tumour cells with the 11q13.3 amplicon during tumorigenesis could be based on the increased doses of one or more of these genes.…”